Yeasts 3000-years-old are alive and other histories of dormant cells

18th August 2019

Translated from the original article in Catalan

A few months ago -April 2019- my friend Jordi Diloli, Professor and Archaeologist, shared a very surprising article (Aouizerat et al 2019) with me. It was echoed on the internet (Borschel-Dan 2019), and I will comment here.

“Resurrected” yeasts from 3,000 years ago

The group of researchers led by Ronen Hazan of the Hebrew University of Jerusalem took samples of 21 clay containers from various sites in present-day Israel from 2500 to 5000 years ago, from the Persian, Philistine and Egyptian (this is the oldest) periods. Archaeologists believed that these vessels contained fermented beverages such as beer or mead (Figure 1). The authors submerged the containers in a rich YPD medium, specific for growing yeasts and other fungi, and incubated them at room temperature for 7 days. Then, samples of this medium were spread on agar plates with the specific medium, and the resulting colonies were isolated for subsequent analyses (Aouizerat et al 2019).

Fig 1 pottery Hazan

Figure 1. Clay vessels from where the yeasts were isolated (Image of Judah Ari Gross, Times of Israel).

 

The isolates that were found were 6 strains of different yeast species, and one of which was Saccharomyces cerevisiae, specifically from a Philistine site dated 3000 years ago. Obviously, it is very surprising that living yeasts of such ancient remains have been isolated. For this reason, the authors of the work carried out a series of experiments that could confirm this unique fact and that the isolates were not a product of contamination.

Firstly Aouizerat et al (2019) showed that it is possible to isolate yeasts from clay vessels that have contained beer or wine after a certain time. They did so with containers with unfiltered beer buried for 3 weeks, and also with another vessel that had repeatedly contained wine but not used last 2 years. With these samples they developed the isolation methodology and in both cases they were able to isolate yeasts. No isolates were obtained from a control sample with filtered beer, therefore without yeasts.

To demonstrate that the isolates were originals of the old vessels because these had contained the fermented beverage, authors applied the same protocol with samples of other ceramics that were surely not for this purpose, and also with sediments near the containers. The result was clearly negative for these samples: only 2 isolated yeasts from 110 samples, while the mentioned 6 yeast strains were isolated from the 21 initial samples. That is, yeasts would be significantly more abundant in containers of alcoholic fermented beverages than in other related archaeological vessels or sediments around them.

Another argument that supports the hypothesis of this work was the identification of these 6 yeasts. Total DNA was obtained and processed to sequence the genomes and compare them with the databases. Two of them, from the Egyptian period, were identified as Saccharomyces delphensis, a species that has been isolated from African dried figs and is not at all common on soil. Therefore, this suggests the use of figs in the alcoholic beverages of these containers. Another isolate was identified as Rhodotorula, common pollutant yeast in African beers. Another was identified as Debaryomyces, a frequent yeast in traditional African sorghum beers. As said before, another isolate was identified as Saccharomyces cerevisiae, the yeast most used to make wine, beer or bread (Figure 2). In spite of this, the genetic sequence of this S. cerevisiae was clearly different from the strains most commonly used today, as commercial or laboratory strains, and therefore the possibility of contamination is excluded. And finally, the other isolate was identified as Hypopichia burtonii, previously isolated yeast from Ethiopian mead.

These genetic data, together with the phenotypic characterization -fermentative kinetics and other biochemical characteristics carried out with the isolates by Aouizerat et al (2019)- suggest that these yeasts actually come from an environment related to alcoholic beverages. These authors even elaborated beer with these isolates and some of them, especially the Saccharomyces, gave a very good analytical and sensory result.

Fig 2 Saccharomyces_cerevisiae_SEM.jpg

Figure 2. Saccharomyces cerevisiae at the scanning electronic microscope (MD Murtey & P Ramasamy)

 

Aouizerat et al (2019) conclude that the isolates are descendants of yeasts that were originally used 3000 years ago, in large quantities and in repeated fermentations. This would have facilitated their survival in pore microenvironments of the ceramic matrix of these containers, and the microcolonies would have continued to grow minimally for millennia thanks to the humidity and residual nutrients. The authors make the analogy with some handmade beers where it is usual that the containers waste serve as starter for new productions.

Finally, the authors of this work speculate that it is possible to isolate microorganisms from archaeological remains, not only yeasts, and that in the case of bacteria it could even be easier, given the resistance characteristics of some of them, such as the sporulated ones.

 

Is there no previous similar work to that of Aouizerat et al (2019) ?

As we have seen, this is certainly a very surprising finding. Scientifically, the work is quite accurate and has been “approved” by the international community: the article is published in an open-access journal with prestige (mBio, high impact factor: 6.7), of the American Society for Microbiology, where all the articles are reviewed by a minimum of two experts, besides the editors. The results presented by the article seem very well worked, and the conclusions are well reasoned.

However, in my opinion it is still almost incredible, and it is strange that nothing like this has been found before. Maybe if someone else had previously tried to isolate such old microorganisms without getting them, perhaps it would not have been published ? Maybe nobody has previously tried to do something similar ? A “malicious” explanation might be that archaeologists have their own interests and microbiologists or molecular biologists have others, and that for this type of work the collaboration of both is needed. Well, it seems not being so, since there are a lot of studies on microorganisms from ancient remains, but they have been almost always focused on the detection and analysis of ancient DNA. These studies demonstrated the presence of certain microorganisms although they did not proceed to isolate them.

 

DNA gives evidence of microorganisms in ancient remains

In relation to yeasts, the oldest evidence is that ribosomal DNA of Saccharomyces cerevisiae has been obtained from residues found in Egyptian wine jars 5000 years old (Cavalieri et al 2003). It must be remembered that the oldest archaeological evidence of large-scale wine production has 7400 years, in north of the Zagros Mountains, in present-day Iran (McGovern et al 1986). As it is known, S. cerevisiae is also the bread and beer yeast, derived from cereals, but since neither S. cerevisiae nor its spores are aerial, surely the use of this yeast in fermented grape juice, as well as dates, figs or honey, historically preceded its use for brewing and bread (Cavalieri et al 2003). It is probable that the wine yeasts naturally occurring in damaged grapes (Mortimer & Polsinelli 1999) were used to ferment other cereal products such as cereals, and after centuries of selection for humans, they evolved into specific strains to ferment food and beverages from cereals.

The genomes of pathogenic microorganisms have also been studied in archaeological remains by means of new massive DNA sequencing techniques, in order to know to epidemic diseases of historical importance, such as black plague, tuberculosis, cholera or leprosy (Andam et al 2016). Logically, in these cases the archaeological remains are human ones, such as bones, teeth, coprolites or mummified tissues. In this way, for example, the phylogeny and evolution of Yersinia pestis strains causing the black plague have been recognized by remains of the Bronze Age (5000 years ago) and until the well-known epidemics of the 6th and 14th centuries (Bos et al 2011). Another well-known case is the Helicobacter pylori genome identified in the intestine of the Ötzi mummy, the iceman in the eastern Alps, 5300 years old (Maixner et al 2016).

DNA has also been isolated from specific bacteria of the human gut, such as Bifidobacterium and Bacteroides, to demonstrate the human presence in archaeological sediments 5000-12000 years old, in north east of Poland (Madeja et al 2009).

It should be remembered that DNA is degraded over time, and in fact it is more unstable than other cellular components. This macromolecule spontaneously suffers damage by oxidation, hydrolysis, and fragmentation in pieces that may be less than 100 bp. Most fossils or other biological remains of more than 100,000 years old no longer contain PCR-amplifiable DNA (Hofreiter et al 2001), although it seems that if the samples are extracted from frozen sediments, with constant temperatures below zero, DNA could be recovered from up to 400,000 years or a little longer (Willerslev et al 2003). In addition the tissues are colonized over time by fungi and bacteria that greatly reduce the relative amount of endogenous molecules and can contribute to giving false positives. The risk of contamination is very high and often this is not taken in account. Generally the DNA of the host that is analysed can be less than 1% of the total DNA found. All these factors complicate the DNA extraction, the construction of sequence libraries, the alignment of DNAs and the analysis of genomes (Andam et al 2016).

Surprisingly, there are a few published works where it is found old DNA of plants, animals and various microorganisms, some million years (My) old, even hundreds of My. The most remarkable are those obtained from amber samples of 20-40 My, and those obtained from a halite 250 My old. This would be comparable to the Jurassic Park fiction where almost non-degraded DNA from the dinosaurs of 100 My old “was recovered”.

Hebsgaard et al (2005) thoroughly reviewed all these more spectacular cases, with the conclusion that these works suffered from inadequate experimental approaches and inadequate authentication of the results. Therefore, there are great doubts as to whether DNA sequences and in some cases viable bacteria could survive such large geological times.

In addition, it is worrying that these works with so old DNA have not been replicated independently in order to confirm their authenticity, and that they did not show a relationship between the age of the sample and the persistence of DNA depending on the different types of bacteria (Willerslev et al 2004). In contrast, Willerslev et al studied the persistence of DNA in permafrost and they found a clear relationship of DNA degradation with time (Figure 3). As seen, DNA amount is very small beyond 100,000 years and it is hardly found beyond 1 My.

Fig 3 willerslev A

Figure 3. Persistence of not degraded bacterial DNA over time (kyr, thousands of years) maintained in permafrost, measured by fluorescence (Willerslev et al 2004).

 

When analysing the bacterial phyla of these DNA, Willerslev et al (2004) observed (Figure 4) that the most persistent are those of Arthrobacter, the main representative of Actinobacteria (high G+C gram-positive), followed by sporulated (Bacillaceae and Clostridiaceae), and finally the Gram-negative Proteobacteria.

Fig 4 willerslev D

Figure 4. Proportions of the main bacterial phyla (Actinobacteria in brown, sporulated in orange and Proteobacteria in blue) based on DNA obtained from permafrost samples, along time (kyr, thousands of years) (Willerslev et al 2004).

 

This increased persistence of non-sporulated Actinobacteria is surprising because sporulated bacteria have always been considered the most resistant of all types of cells. Although endospores have special adaptations such as proteins binding DNA to reduce the rate of genetic modifications, they do not have active metabolism or repair and their DNA will degrade over time. The mechanism of greater resistance of Actinobacteria is unknown, but there may be some activity and repair of DNA at temperatures below zero, and/or adaptations related to the dormant cells state (Willerslev et al 2004).

Anyway, the limit for PCR-amplifying the DNA would be between 400,000 years and 1.5 My for samples kept below zero, but this is much more unlikely in non-frozen materials, such as the amber of halite samples of million years, and much less likely to find viable cells from these samples so old (Willerslev et al 2004).

 

“Resurrected” bacteria

The same commented works where DNA of some millions of years (My) was found, are the most surprising cases of having “resurrected” microorganisms, basically bacteria: viable cells of the sporulated Bacillus from amber samples of 30 My (Cano & Borucki 1995), Staphylococcus also from amber of about 30 My (Lambert et al 1998), and the most spectacular case of Bacillus from an halite of 250 My (Vreeland et al 2000 ). This sporulated bacterium would have been in a hyper-saline environment of the last Permian and trapped in a salt crystal, surviving until now. In the case of Staphylococcus isolated from amber, in spite of not being sporulated, they are bacteria very resistant to extreme conditions, and which have been isolated also from ancient permafrost and very dry environments (Lambert et al 1998).

In spite of this, the revision of these cases by Hebsgaard et al (2005) concludes that none of them fulfilled the relative rate of molecular distance test, which is the probable rate of mutations calculated in comparison to related lineages. Therefore, these isolations are arguable and not reproduced. In addition, in the case of the 250 My Bacillus, it has been argued that the inclusion of bacteria in the halite could be the consequence of a subsequent recrystallization (Lowenstein et al 2011).

Another review on microorganism preservation records (Kennedy et al 1994) comments published cases up to 600 My, indicating that it is curious that there are several cases with more than 1 My, and also cases with less than 10,000 years ago, but there are very few cases of intermediate periods. These authors also point out the doubts raised by works with surviving bacteria so old, which would surely be artefacts or contaminations.

On the other hand, the most credible works are those of Abyzov et al (2006) and Soina et al (2004), which demonstrated the presence of several living microorganisms, both prokaryotes and eukaryotes (especially yeasts, but also some microalgae), in Antarctic ice samples that have some thousands of years. These authors combined classical microbiological methods, such as enrichment and isolation of colonies, together with epifluorescence microscopy, electronic microscopy, and molecular techniques. The bacteria found were Gram-positive (Micrococcus) and gram-negative (Arthrobacter), which are not sporulated, but they have cist-shaped dormant cells, which can survive while maintaining viability at temperatures below 0ºC for some thousands of years.

When geologically ancient DNA findings are published as well as viable cultures of ancient samples, the independent reproduction of the results by another laboratory is fundamental, to exclude any contamination from the same laboratory. In the case of having recovered living cells, it is necessary to demonstrate the reproducibility of the isolation, sequencing the genomes of the cultures obtained in independent laboratories from the same sample, and checking that in both cases the genomes coincide (Hebsgaard et al 2005).

From the remains of the Roman fort of Vindolanda, in the north of England, viable endospores of Thermoactinomyces, member of Bacillales (Unsworth et al 1977) have been recovered. They are about 1900 years old and the remains were a mixture of clay with straw and other vegetable materials. The authors propose to use these sporulated bacteria as indicators in archaeological studies.

Besides sporulated bacteria, there are several groups of non-sporulated ones for which anabiosis resistance abilities have been demonstrated. In particular, they have been isolated from permafrost and the tundra soil of Siberia of about 1 My (Suzina et al., 2006), in the limit of what we mentioned earlier (Willerslev et al 2004), which is quite difficult to believe. In order to study experimentally the formation of these anabiosis forms, Suzina et al incubated several gram-positive and gram-negative bacteria, and some archaea, in poor media with limiting nitrogen, and after a few months they obtained their dormant cells. They had cist structures, with capsule and a thickened cell wall, intramembranous particles and a condensed nucleoid (Figure 5). They also observed that these cysts did not have metabolic activity and supported stress factors such as lack of nutrients or heating.

Studying the permafrost isolates, they confirmed that there are cist structures very similar to those obtained in the laboratory, with multi-layer wall structures of up to 0.4 μm. In fact, these authors believe that most of the bacteria present in the permafrost and the tundra are in the form of a cyst (Suzina et al 2006).

Fig 5 fig2 modi Suzina

Figure 5. Sections of a vegetative cell (a) of Micrococcus luteus and of a cyst cell (b) of the same bacterium, obtained after 9 months of culture in a medium limiting in nitrogen. C, microcapsule; CW, cell wall; OL1, 2, 3, outer layers of the cell wall; IL, inner layer of the wall; CM, cytoplasmic membrane; N, nucleoid. The bar measures 0.3 μm (Suzina et al., 2006).

 

Other “resurrected” yeasts and fungi

Besides the surprising mentioned article by Aouizerat et al. (2019), there are other few published cases of yeasts and other “resurrected” fungi such as the following.

Chicha is a beer-like beverage from corn, yellowish and slightly effervescent, elaborated and consumed by Andean populations for some thousands of years, whose traditional process has the peculiarity of using amylase of saliva for convert the starch into fermentable sugars. Fermentation traditionally took place in clay containers called “pondos”. From the remains of the chicha pondos from the Hipia culture in Quito (2100-2800 years old), various yeast were isolated, especially Candida, Pichia and Cryptococcus (Gomes et al 2009). Interestingly, some of these yeasts have been confirmed molecularly as Candida theae, similar to those isolated from contaminated Asian tea (Chang et al 2012). It is worth mentioning the absence of Saccharomyces in these ancient chicha, although today it is the main yeast, coming probably from beer and wine fermentation that led the Spaniards (Gomes et al 2009).

From Greenland ice samples of about 100,000 years (Ma et al 1999), several microorganisms were revived, such as bacteria (Micrococcus, RhodotorulaSarcina) and yeasts (Candida, Cryptococcus) and other fungi (PenicilliumAspergillus). The authors also isolated the DNAs and demonstrated the phylogenetic relationship of the isolates. Once again, we see how ice provides a stable environment that facilitates the conservation of microorganisms and their DNA.

Raghukumar et al (2004) have recovered living Aspergillus (sporulated Ascomycota fungus) and other fungi from sediment samples of the deep-sea, about 5900 m deep in the Chagos trench, south of the Maldives, in the Indian Ocean. Based on the depth in the sediment and the present Radiolaria, authors estimated that they correspond to a minimum of 180,000 years, and up to 430,000 years in some samples. From the isolates identified as A. sydowii they obtained spores that germinated and grew in hydrostatic pressure equivalent to the depth of 5000 m, and at a temperature of 5ºC. With microscopy of epifluorescence and bright field, the fungal hypha and their relation to the particles of the sediment are clearly observed (Figure 6). It seems that this Aspergillus found in the deep-sea is the oldest fungus recovered alive so far. The authors suggest that preservation would have been possible thanks to high hydrostatic pressure, along with low temperature.

Fig 6 Raghukumar Aspergillus deepsea indian

Figure 6. Photomicrographies of deep-sea sediment (5900 m) of the Indian Ocean with hyphae of Aspergillus sydowii and sediment particles. (a) epifluorescence microscopy combined with that of bright field; (b) epifluorescence (Raghukumar et al 2004).

 

One of the most surprising works, and hard to believe, is that of Kochkina et al (2001), where a lot of fungi of all kinds and bacteria, especially actinobacteria, were isolated from samples of permafrost from Russia, Canada and Antarctica reaching 3 My old. The authors even suggested that there is no limit of years to recover viable microorganisms. This article has had very little echo, and it is not even mentioned by later articles as Raghukumar et al. (2004).

 

Conclusions

As we have seen, evidence of DNA from no-living yeasts in ancient remains related to winemaking dates back to around 5000 years in ancient Egypt (Cavalieri et al 2003). Regarding other microorganisms, taking into account the natural degradation of DNA over time, it seems that the oldest samples would be about 400,000 years at most, in particular actinobacteria in frozen sediments such as permafrost (Willerslev et al 2003 ). Publications of bacterial DNA recovered from several millions years (up to 600 My) have many scientific concerns about their credibility and reliability (Kennedy et al 1994).

With regard to living yeast as those of 3000 years apparently isolated by Aouizerat et al (2019), it seems that Candida and others were isolated from containers to elaborate chicha about 2800 years old (Gomes et al 2009), although this reference is a review and the original work does not appear to have been published. Other authors (Abyzov et al 2006; Soina et al 2004) also find alive yeasts, without specifying which ones, in Antarctic ice samples of some thousands of years. More surprising are the isolated isolations of yeast and other fungi and bacteria from Greenland ice samples 100,000 years old (Ma et al 1999), as well as those of Aspergillus from the Indian Ocean seabed of about 180,000 years (Raghukumar et to 2004).

Regarding other “resurrected” microorganisms, some of the most reliable are the several Antarctic ice bacteria of some thousands of years (Abyzov et al 2006) and Thermoactinomyces spores of Roman remains 1900 years old (Unsworth et to 1977). Of the oldest, perhaps the anabiosis forms of bacteria conserved in permafrost a million years old (Suzina et al., 2006) would have certain likelihood. Curiously, these bacteria would be non-sporulated but they would have a cyst structure, with multi-layer walls and other intracellular modifications. The other findings of “resurrected” bacteria from more millions of years of amber or halite, just like their DNA and also because of this, are very hard to believe (Hebsgaard et al 2005).

Thinking in the cellular forms of resistance and anabiosis, as the bacterial endospores and the mentioned cysts, it must be remembered that yeasts, like many other fungi, have the ability to produce spores, in particular ascospores as they are Ascomycetes. Although these ascospores have a greater capacity for resistance than vegetative cells in dry conditions or other inhospitable environments and have a persistence in time, apparently there is no work (or I have not been found) related to the recovery of yeasts ascospores from ancient remains.

The work of Aouizerat et al (2019) makes no mention of the yeast spores, neither as a possible explanation of the yeast survival in these ancient remains. In fact, they propose that the microcolonies of yeasts on ceramics pores would have continued to grow minimally during these 3000 years thanks to the humidity and residual nutrients. Well, we do not know, and neither if the yeast ascospores have had any role.

Finally, we can believe the finding of Aouizerat et al (2019) is truth, but obviously further investigation in other similar archaeological samples must be done. This research should be done not only for yeasts, but also for bacteria of other fermented products. Besides considering the sporulated ones, other bacteria should be considered, that could survive thanks to the cell cysts or other forms of anabiosis.

 

Bibliography

Abyzov SS et al (2006) Super-long anabiosis of ancient microorganisms in ice and terrestrial models for development of methods to search for life on Mars, Europa and other planetary bodies. Adv Space Res 38, 1191-1197

Andam CP et al (2016) Microbial genomics of ancient plagues and outbreaks. Trends Microbiol 24, 978 –990

Aouizerat T et al (2019) Isolation and characterization of live yeast cells from ancient vessels as a tool on bio-archaeology. mBio 10, 2, 1-21

Borschel-Dan A (2019) Israeli scientists brew groundbreaking “ancient beer” from 5,000-year-old yeast. The Times of Israel, 22nd may 2019.

Bos KI et al (2011) A draft genome of Yersinia pestis from victims of the Black Death. Nature 478, 506–510

Cano, R.J. and Borucki, M.K. (1995) Revival and identification of bacterial spores in 25- to 40-million year-old Dominican amber. Science 268, 1060–1064

Cavalieri D et al (2003) Evidence for S. cerevisiae fermentation in ancient wine. J Mol Evol 57:S226-232

Chang CF et al (2012) Candida theae sp. nov., a new anamorphic beverage-associated member of the Lodderomyces clade. Int J Food Microbiol 153, 10-14.

Gomes FCO et al (2009) Traditional foods and beverages from South America: microbial communities and production strategies. Chapter 3 in Industrial Fermentation, ed. J Krause & O Fleischer, Nova Science Publishers.

Hofreiter M et al (2001) Ancient DNA. Nature Rev Genet 2, 353–359.

Kennedy MJ et al (1994) Preservation records of micro-organisms: evidence of the tenacity of life. Microbiology 140, 2513-2529.

Kochkina GA et al (2001) Survival of micromycetes and actinobacteria under conditions of long-term natural cryopreservation. Microbiology 70, 356-364

Lambert LH et al (1998) Staphylococcus succinus sp. nov., isolated from Dominican amber. Int J Syst Bacteriol 48, 511-518

Lowenstein TK et al (2011) Microbial communities in fluid inclusions and long-term survival in halite. GSA Today 21, 4-9

Ma L et al (1999) Revival and characterization of fungi from ancient polar ice. Mycologist 13, 70-73.

Madeja J et al (2009) Bacterial ancient DNA as an indicator of human presence in the past: its correlation with palynological and archaeological data. J Quaternary Sci 24, 317-321.

Maixner F et al. (2016) The 5300-year-old Helicobacter pylori genome of the Iceman. Science 351, 162–165

McGovern PE et al (1986) Neolithic resinated wine. Nature 381:480–481

Mortimer R & M Polsinelli (1999) On the origins of wine yeast. Res Microbiol 150, 199-204

Raghukumar C et al (2004) Buried in time: culturable fungi in a deep-sea sediment core from the Chagos Trench, Indian Ocean. Deep Sea Res Part I: Oceanog Res Papers 51, 1759-1768

Soina VS et al (2004) The structure of resting microbial populations in soil and subsoil permafrost. Astrobiology 4 (3), 345–358.

Suzina et al (2006) The structural bases of long-term anabiosis in non-spore-forming bacteria. Adv Space Res 38, 1209-1219.

Unsworth BA et al (1977) The Longevity of Thermoactinomycete Endospores in Natural Substrates. J Appl Microbiol 42, 45-52

Vreeland RH et al (2000) Isolation of a 250 milion-year-old halotolerant bacterium from a primary salt cristal. Nature 407, 897-900.

Willerslev E et al (2003) Diverse plant and animal DNA from Holocene and Pleistocene sedimentary records. Science 300, 791-795

Willerslev E et al (2004) Long-term persistence of bacterial DNA. Curr Biol 14, PR9-R10.

 

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About Albert Bordons

Professor at "Universitat Rovira i Virgili" in Tarragona. Born in Barcelona 1951. Scientific areas: microbiology, biochemistry, biotechnology, oenology. I like: nature, biological sciences, photography, mountains, ... Languages: catalan (first one), spanish, french, english and some italian.

Posted on 18/08/2019, in Bacteria, Genetics and molecular biology, Wines and Oenology and tagged , , , , , , , , , , , , , , , , , , , , , . Bookmark the permalink. 1 Comment.

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